EFFECTS OF SHIGA TOXIN 2 IN RATS IN EARLY STAGE OF PREGNANCY

SACERDOTI FLAVIA; AMARAL MARIA MARTA; ZOTTA ELSA; IBARRA CRISTINA

SANTOS-SAN PABLO-BRASIL

Congreso; XXI ALAM CONGRESO LATINOAMERICANO DE MICROBIOLOGIA; 2012

Sociedad Latinoamericana de Microbiología

Introduction: Shiga toxin (Stx) producing Escherichia coli (STEC) causes diarrhea and hemorrhagic colitis, and is the leading cause of hemolytic uremic syndrome (HUS), a systemic complication that is attributed to the expression of Shiga toxins type 1 (Stx1) and type 2 (Stx2) and its variants1. Argentina has the highest rate of HUS in the world. Over the last 10 years, approximately 500 HUS cases were reported annually. The incidence has ranged between 7.8 and 17 cases per 100,000 children less than 5 year of age and the lethality was between 2 and 5%. This rate is 10-fold higher than in other industrialized countries. In some studies, evidence of STEC infection was found in around 60% of Argentinian HUS cases, and E. coli O157 was the predominant serogroup isolated2. The recent outbreak of HUS caused by Stx2 in Germany affected in high number adults and particularly women3. Different studies have demonstrated the role of ruminants, mainly cattle, as STEC reservoir and foods as vehicle of transmission4. Stx is the major virulence determinant of STEC and it has been shown to be directly toxic to kidney and brain through the receptor globotriaosylceramide (Gb3)5. Maternal infections have been linked to several adverse pregnancy outcomes in humans, including fetal anomalies, intrauterine fetal death, premature delivery, premature rupture of the membranes and abortion. Previous works in our laboratory have shown premature delivery of dead fetuses in rats intraperitoneally (i.p) injected with supernatant from recombinant E. coli containing Stx2 and lipopolysaccharide (LPS) 6-8. We have proposed that Stx2 could be one of the causes not yet investigated of feto-maternal morbimortality. In the present work we have analyzed the effects of Stx2 in rats in the early stage of pregnancy. Materials and Methods: Timed pregnant rats Sprague Dawley (n= 4-6) were i.p. injected with 200 µl of 0.25, 0.5 or 1 ng Stx2 (Phoenix Lab, USA) /g of body weight (bwt) on day 8 of gestation (gd 8). Control rats were injected with the same volume of phosphate-buffered saline (PBS). Rats were controlled every 24 h until the day of delivery. Body weight, water and food intake were registered up to 10 days post injection. Because of bwt variability data are presented as Δbwt, where Δbwt = bwt on day post injection ? bwt on day of injection. To observe fetus status, some groups of rats were sacrificed under CO2 anesthesia at different times after the treatment. Gestational sacs were opened and the fetoplacental resorption was evaluated macroscopically. In addition, fetomaternal tissues and kidney were removed, fixed, dehydrated and embedded in paraffin. Sections of 5 µm were mounted on 2% silane-coated slides, stained with hematoxylin?eosin and observed by light microscopy. To determine the presence of the receptor Gb3 in the feto-maternal unit, total lipids were extracted and a thin layer chromatography (TLC) was performed. Statistical analysis was done using the Graph Pad Prism Software. Comparisons between values of different groups were performed using one-way ANOVA and significances were determined using Bonferroni test. Results: All the experimental mothers showed a significant decrease of food intake after Stx2 treatment (+p